WO2007135118A1

WO2007135118A1 - Soluble, stable insulin-containing formulations

Info

Publication number: WO2007135118A1
Application number: PCT/EP2007/054863
Authority: WO
Inventors: Helle Birk Olsen; Niels Christian Kaarsholm; Palle Jakobsen; Per Balschmidt
Original assignee: Novo Nordisk A/S
Priority date: 2006-05-24
Filing date: 2007-05-21
Publication date: 2007-11-29

Abstract

The present invention relates to pharmaceutical formulations comprising acid stabi- lized insulin, and a salt of protamine or other positively charged peptides, to methods of pre- paring such formulations, and to uses of such formulations in the treatment of diseases and conditions for which use of the insulin peptide(s) contained in such formulations is indicated.

Description

SOLUBLE, STABLE INSULIN-CONTAINING FORMULATIONS

FIELD OF THE INVENTION

The present invention relates to pharmaceutical formulations comprising acid stabilized insulin and a salt of protamine or other positively charged peptides, to methods of pre- paring such formulations, and to uses of such formulations in the treatment of diseases and conditions for which use of the insulin peptide(s) contained in such formulations is indicated.

BACKGROUND OF THE INVENTION

Diabetes mellitus is a metabolic disorder in which the ability to utilize glucose is more or less completely lost. About 2% of all people suffer from diabetes. Since the discovery of insulin in the 1920's, continuous strides have been made to improve the treatment of diabetes mellitus. To help avoid extreme glucose levels, diabetic patients often practice insulin replacement therapy, whereby insulin is administered by injection.

In the treatment of diabetes mellitus, many varieties of insulin compositions have been suggested and used, including regular insulin, Semilente^® insulin, isophane insulin, insulin zinc suspensions, protamine zinc insulin, and Ultralente^® insulin. As diabetic patients typically are treated with insulin for several decades, there is a major need for safe and life quality improving insulin compositions. Some of the commercially available insulin compositions are characterized by a fast onset of action, while other compositions have a relatively slow onset but show a more or less prolonged action. Fast acting insulin compositions are usually solutions of insulin, while retarded acting insulin compositions can be suspensions containing insulin in crystalline and/or amorphous form precipitated by addition of zinc salts alone or by addition of protamine or by a combination of both. In addition, some patients use compositions having both a fast onset of action and a more prolonged action. Such a composition may be an insulin solution wherein protamine insulin crystals are suspended. Some patients prepare the final composition them- selves by mixing an insulin solution with a suspension composition in the desired ratio.

Human insulin consists of two polypeptide chains, the so-called A and B chains, which contain 21 and 30 amino acid residues, respectively. The A and B chains are interconnected by two cystine disulphide bridges and a third disulfide bridge is intra A chain. Insulin from most other species has a similar construction, but may not contain the same amino acid residues at corresponding positions.

The development of genetic engineering has made it possible to easily prepare a great variety of insulin compounds analogous to human insulin. In these insulin analogs, one or more of the amino acid residues have been substituted with other amino acid residues which can be coded for by the nucleotide sequences. Since human insulin, as explained above, contains 51 amino acid residues, it is obvious that a large number of insulin analogs is possible, and a great variety of analogs with interesting properties have been prepared. In human insulin solutions with a concentration of interest for injectable compositions, the insulin molecule is present in associated form as a hexamer (Brange et al. Diabetes Care 13, (1990), 923 - 954). After subcuta- neous injection, it is believed that the rate of absorption by the blood stream is dependent on the size of the molecule, and it has been found that insulin analogues with amino acid residue substitutions which counteract or inhibit this hexamer formation have an unusually fast onset of action (Brange et al.: Ibid). This can be of great therapeutic value for the diabetic patient.

A general survey of pharmaceutical compositions containing insulin is given by Brange et al. in Galenics of Insulin, Springer-Verlag (Berlin, 1987).

Scott and Fisher (1936) disclose suspensions of insulin containing 1 mM protamine sulphate and 0.15 mM insulin at pH 7.2.

Pharmaceutical compositions which are based on analogues of human insulin have e.g. been presented by Heinemann et al., Lutterman et al. and Wiefels et al. at the "Frontiers in Insulin Pharmacology" International Symposium in Hamburg, 1992.

US 5474 978 (EIi Lilly) discloses a rapidly acting parenteral formulation comprising a human insulin analogue hexamer complex consisting of six monomeric insulin analogues, zinc ions and at least three molecules of a phenolic derivative.

Normally, insulin compositions are administered by subcutaneous injection. What is important for the patient is the profile of action of the insulin composition, i.e. the action of insulin on the glucose metabolism as a function of the time from the injection, including the time for the onset of insulin action, the maximum value and the total duration of action. A variety of insulin compositions with different profiles of action are required by patients. An individual patient may thus on the same day use insulin compositions with very different profiles of action. The profile of action required for any given patient at any given time depends upon several factors, e.g. the time of the day and the amount and composition of any meal eaten by the patient.

Also important for the patient is the chemical stability of the insulin compositions, especially due to the abundant use of pen-like injection devices such as devices which contain Pen- fill^® cartridges, in which an insulin composition is stored until the entire cartridge is empty. This may last 1 to 2 weeks or more for devices containing a 1.5 or 3.0 ml cartridge. During storage, covalent chemical changes in the insulin structure occur. This may lead to the formation of molecules which are less active and potentially immunogenic such as deamidation products and higher molecular weight transformation products (dimers, polymers, etc.). A comprehensive study on the chemical stability of insulin is given by Jens Brange in "Stability of Insulin", Kluwer Academic Publishers, 1994. Compositions comprising insulin and insulin analogues are traditionally formulated using various additives, for example sodium phosphate (buffer), Zn²⁺ (stabilizer), phenol/m- cresol (preservative and stabilizer), sodium chloride (isotonicity agent and stabilizer), and glycerol/mannitol (isotonicity agents). The shelf-life of insulin products is mainly compromised by the formation of soluble aggregates (dimers and polymers) over time, despite the fact that insulin is typically stored at a low temperature of no more than about 5°C, which improves the shelf-life considerably compared to storage e.g. at room temperature. In addition, insulin products are subject to the formation of insoluble aggregates (fibrils) as a result of shaking, e.g. when carried in the pocket of a patient or during transport. It is essential for the quality of an insulin product that the tendency to form such soluble and insoluble aggregates as a result of chemical or physical influences is reduced to an absolute minimum.

Acta Pharmaceutics Nordica 4(4), 1992, pp. 149-158 discloses insulin compositions with a sodium chloride concentration in the range of 0 to 250 mM. The major part of the compo- sitions, including those which additionally comprise glycerol, contain a rather high amount of sodium chloride, i.e. 0.7%, corresponding approximately to a concentration of 120 mM.

US Patent No. 5,866,538 (Novo Nordisk) discloses insulin compositions having improved chemical stability, the compositions comprising human insulin or an analog or derivative thereof, glycerol and/or mannitol and 5-100 mM of a halogenide, e.g. sodium chloride. US Patent No. 6, 174,856 (Novo Nordisk) discloses stabilized aqueous compositions comprising human insulin or an analog or derivative thereof, a buffer selected from glycylgly- cine, citrate or TRIS and metal ions, in particular, calcium or magnesium ions.

US Patent No. 6,451 ,762 (Novo Nordisk) discloses protracted acting water soluble aggregates of derivatives of human insulin. US Patent No. 6,551 ,992 (EIi Lilly) discloses monomeric insulin analog formulations stabilized against aggregation in which the buffering agent is either TRIS or arginine.

US Patent No. 5,747,642 (EIi Lilly) discloses parenteral pharmaceutical formulations which comprise a monomeric insulin analog, zinc, protamine and a phenolic derivative.

US Patent No. 6,465,426 (EIi Lilly) discloses insoluble compositions comprising an acylated insulin or acyalted insulin analog complexed with zinc, protamine and a phenolic compound such that the resulting microcrystal is analogous to the neutral protamine Hage- dorn (NPH) insulin crystal form.

Although progress has been made in the chemical and physical stabilization of insulin-containing compositions, the need still remains for soluble, stable formulations of insulin or analogs or derivatives thereof, or mixtures of the foregoing, that exhibit a prolonged action profile upon administration in vivo. SUMMARY OF THE INVENTION

The present application discloses that formulations containing certain salts of protamine or other positively charged peptides at certain concentrations allow soluble stable preparations of acid stabilized insulin, to be formulated at pHs below 5.5. The present formu- lations are also physically and chemically stable at a pH below 5.5 and exhibit a prolonged profile of action thus rendering them shelf-stable and suitable for invasive (e.g. injection, subcutaneous injection, intramuscular, intraveneous or infusion ) as well as non-invasive (e.g. nasal, oral, pulmonary, transdermal or transmucosal e.g. buccal ) means of administration. The present invention therefore relates to pharmaceutical formulations comprising acid stabilized insulin and a salt of protamine or other positively charged peptides where the salt is present in a concentration of at least 0.01 mM and the pH of the formulation is less than about 5.0. The pharmaceutical formulations of the invention may further contain at least one of the following components: a preservative, a divalent metal ion such as zinc cobalt, magnesium or calcium or combinations of these ions, an isotonicity agent, a buffer and a surfactant.

The present invention further relates to methods of treatment using the pharmaceutical formulations of the invention where the compositions are administered in an amount effective to combat the disease, condition, or disorder for which administration of the insulin peptide contained in the formulation is indicated. In one embodiment, the formulations of the invention may be used in the treatment of type 1 and type 2 diabetes.

The present invention also relates to methods for producing the pharmaceutical formulations of the invention.

In one embodiment, the method for preparing formulations of the invention com- prises: a) preparing a solution by dissolving a divalent metal ion in water or buffer; b) preparing a solution by dissolving the preservative in water or buffer; c) preparing a solution by dissolving the isotonicity agent in water or buffer ; d) preparing a solution by dissolving the surfactant in water or buffer; e) preparing a solution by dissolving the insulin, insulin analog, insulin derivative or a mixture of the foregoing in water or buffer; f) preparing a solution by dissolving a salt of protamine or other positively charged peptides in buffer or water; g) mixing solution e) and one or more of solutions a), b), c), and d); h) mixing solution g) with solution f); and i) adjusting the pH of the mixture in h)l to the desired pH of less than 5.0. BRIEF DESCRIPTION OF THE FIGURES

Testing the solubility of insulin at pH=7.4 in the presence of protamine acetate

Figure 1 show the solubility of human insulin in the presence of 1.2mM and 2.OmM concentrations of the acetate salt of R12. The preparations are 0.4mM human insulin, 25mM tricresol, 1.6% glycerol, 40 ppm tween-20 and R12 added as noted in the legends.

DESCRIPTION OF THE INVENTION

The pharmaceutical formulations of the invention comprise acid stabilized insulin and a salt of protamine or other positively charged peptides where the salt is present in a concentration of at least 0.01 mM and the pH of the formulation is less than about 5.5.

The pharmaceutical formulations of the invention are chemically stable and soluble at pHs less than 5.0. By "soluble at a given pH" is meant that the insulin contained in the formulation of the invention is fully dissolved at the pH of the formulation where methods for determining whether the insulin contained in the formulation of the invention is dissolved are known in the art.

In one embodiment, the pharmaceutical formulation may be subjected to centrifugation for 20 minutes at 30,000 g and then the insulin concentration in the supernatant may be determined by RP-HPLC. If this concentration is equal within experimental error to the insulin concentration originally used to make the formulation, then the insulin is fully soluble in the formula- tion of the invention.

In another embodiment, the solubility of the insulin peptide(s) in a formulation of the invention can simply be determined by examining by eye the container in which the formulation is contained. Insulin is soluble if the solution is clear to the eye and no particulate matter is either suspended or precipitated on the sides/bottom of the container. Of course, it is to be understood by the skilled artisan that the solubility of insulin in a formulation of the invention may be affected not only by the composition of the formulation and its pH but also by the temperature and time at which the formulation is stored prior to measurement of solubility.

In the formulations of the invention, the term "acid stabilized insulin" as used herein re- fers to an analogue of human insulin that has reduced tendency to deamidate or dimerize at pH values below 7 compared to human insulin. Specifically, the analogue cannot have Asn or Asp as a C-terminal residue, that is an acid stabilized insulin always has a substitution of Asn in position A21 with any other amino acid with the exception of Asp.

In one embodiment the acid stabilized insulin is an analogue of human insulin wherein position B28 is Asp or GIu. In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B28 is Asp.

In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B28 is GIu. In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B29 is Pro, Asp or GIu.

In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B29 is Pro or GIu.

In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B29 is Pro.

In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B29 is GIu.

In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B28 is Asp or Lys, and position B29 is Lys or Pro. In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B9 is Asp or GIu.

In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B10 is Asp or GIu or GIn.

In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B10 is GIu.

In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B10 is GIn.

In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B1 is GIy. In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B3 is Lys, Thr, Ser, Ala or GIn.

In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B3 is Lys, Thr, Ser or Ala.

In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B3 is Lys or Ala.

In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B3 is Lys.

In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B3 is Lys and position B29 is GIu. In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B25 is deleted. In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B27 is deleted.

In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position B30 is deleted. In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position A18 is GIn.

In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position A21 is Ala, Arg, GIn, GIu, GIy, His, lie, Leu, Met, Phe, Ser, Thr, Trp, Tyr, VaI or hSer. In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position A21 is Ala, Arg, GIn, GIy, lie, Leu, Phe, Ser, Thr, VaI or hSer.

In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position A21 is Ala or GIy.

In another embodiment the acid stabilized insulin is an analogue of human insulin wherein position A21 is GIy.

In another embodiment the acid stabilized insulin is a derivative of human insulin or an analogue thereof having one or more lipophilic substituents.

In another embodiment the acid stabilized insulin is a derivative of human insulin or an analogue thereof wherein the N^ε-amino group in position B29Lys is modified by covalent acylation with a hydrophobic moiety such as a fatty acid derivative or an litocholic acid derivative.

In another embodiment the acid stabilized insulin derivative is selected from the group consisting of B29N^ε-hexadecandioyl-γ-Glu desB30 insulin, B29-N^ε-myristoyl-des(B30) human insulin, B29-N^ε-palmitoyl-des(B30) human insulin, B29-N^ε-myristoyl human insulin, B29-N^ε-palmitoyl human insulin, B28-N^ε-myristoyl Lys^B28 Pro⁸²⁹ human insulin, B28-N^ε- palmitoyl Lys^B28 Pro^B29 human insulin, B30-N^ε-myristoyl-Thr^B29Lys^B30 human insulin, B30-N^ε- palmitoyl-Thr^B29Lys^B30 human insulin, B29-N^ε-(N-palmitoyl-γ-glutamyl)-des(B30) human insulin, B29-N^ε-(N-lithocholyl-γ-glutamyl)-des(B30) human insulin, B29-N^ε-(ω-carboxyheptadecan- oyl)-des(B30) human insulin and B29-N^ε-(ω-carboxyheptadecanoyl) human insulin all of these with an acid stabilizing mutation in position A21.

In another embodiment, the acid stabilized analogs of human insulin contain any combination of additional stabilizing substitutions.

In another embodiment, the acid stabilized analogs of human insulin contain any combination of the additional stabilizing substitutions in positions B1 , B3, A18. In another embodiment the acid stabilized insulin is an analogue of human insulin selected from the group consisting of: In another embodiment the insulin is an analogue of human insulin selected from the group consisting of:

A21G

A21Q, A21A,

A21T

A21 R

All of the combinations below could be A21 Q/A/T/R as well:

A21G, B28K, B29P A21G, B28D

A21G, B28E

A21G, B3K, B29E

A21G, desB27

A21G, B9E A21G, B9D

A21G, B10E

A21G, desB25

A21G, B25H

A21G, desB30 A21G, B28K, B29P

A21G, B28K, B29P, desB30

A21G, B28D, desB30

A21G, B28E

A21G, B28E, desB30 A21G, B3K, B29E

A21G, B3K, B29E, desB30

A21G, desB27, desB30

A21G, B9E/D

A21G, B9E, desB30 A21G, B9D, desB30

A21G, B10E/D/Q

A21G, B10E, desB30

A21G, B10Q, desB30

A21G, desB25, desB30. In another embodiment the insulin is an analogue of human insulin selected from the group consisting of: B1 G, A21G

B1G, A21G, B28K, B29P

B1G, A21G, B28D

B1G, A21G, B28E B1G, A21G, B3K, B29E

B1G, A21G, desB27

B1G, A21G, B9E

B1G, A21G, B9D

B1G, A21G, B10E B1G, A21G, B10Q

B1G, A21G, desB25

B1G, A21G, B25H

B1G, A21G, desB30

B1G, A21G, B28K, B29P B1G, A21G, B28K, B29P, desB30

B1G, A21G, B28D, desB30

B1G, A21G, B28E

B1 G, A21G, B28E, desB30

B1 G, A21G, B3K, B29E B1 G, A21G, B3K, B29E, desB30

B1 G, A21G, desB27, desB30

B1 G, A21G, B9E/D

B1 G, A21G, B9E, desB30

B1 G, A21G, B9D, desB30 B1 G, A21G, B10E/D/Q

B1 G, A21G, B10E, desB30

B1 G, A21G, B10Q, desB30

B1 G, A21G, desB25, desB30,

B1 G, A21G,B25H, desB30 In another embodiment, the acid stabilized insulin is an analogue of human insulin from above three lists further modified in positions B3 and A18, eg B3T, B3S, B3Q and

A18Q.

In another embodiment, the acid stabilized insulin is an analogue of human insulin from the above three lists further modified as follows: B3T, B28D

B3T, desB27. In another embodiment, the acid stabilized insulin is an analogue of human insulin from the above three lists further modified by deletion of B30.

In another embodiment, the insulin analogs and derivatives are selected from among those disclosed in EP 0 792 290 (Novo Nordisk MS), EP 0 214 826 and EP 0 705 275 (Novo Nordisk MS), US 5,504,188 (EIi Lilly), EP 0 368 187 (Aventis), US patents

5,750,497 and 6,011 ,007, EP 375437 and EP 383472 and where such insulins may include, but are not limited to, Lys^B29 (N^ε-tetradecanoyl) des(B30) human insulin, Lys^B29-(N^ε-(γ- glutamyl-N^α-lithocholyl) des(B30) human insulin, N^εB29-octanoyl insulin, insulin glargine (insulin glargine, also known as Lantus®, differs from human insulin in that the amino acid aspar- agine at position A21 is replaced by glycine and two arginines are added to the C-terminus of the B-chain), insulin glulisine ( insulin glulisine, also known as Apidra®, differs from human insulin in that the amino acid asparagine at position B3 is replaced by lysine and the lysine in position B29 is replaced by glutamic acid) Lys^B28 Pro⁸²⁹ human insulin (Humalog®) , Asp^B28 human insulin, or insulin aspart (Novolog®) insulin all of these with an acid stabilizing muta- tion in position A21.

In one embodiment, the acid stabilized insulin is a derivative of human insulin or a human insulin analogue where the derivative contains at least one lysine residue and a lipophilic substituent is attached to the epsilon amino group of the lysine residue.

In one embodiment, the lysine residue to which the lipophilic substituent is attached is present at position B28 of the insulin peptide.

In another embodiment, the lysine residue to which the lipophilic substituent is attached is present at position B29 of the insulin peptide.

In yet another embodiment, lipophilic substituent is an acyl group corresponding to a carboxylic acid having at least 6 carbon atoms. In another embodiment, the lipophilic substituent is an acyl group, branched or unbranched, which corresponds to a carboxylic acid having a chain of carbon atoms 8 to 24 atoms long.

In another embodiment, the lipophilic substituent is an acyl group corresponding to a fatty acid having at least 6 carbon atoms. In another embodiment, the lipophilic substituent is an acyl group corresponding to a linear, saturated carboxylic acid having from 6 to 24 carbon atoms.

In another embodiment, the lipophilic substituent is an acyl group corresponding to a linear, saturated carboxylic acid having from 8 to 12 carbon atoms.

In another embodiment, the lipophilic substituent is an acyl group corresponding to a linear, saturated carboxylic acid having from 10 to 16 carbon atoms. In another embodiment, the lipophilic substituent is an oligo oxyethylene group comprising up to 10, preferably up to 5, oxyethylene units.

In another embodiment, the lipophilic substituent is an oligo oxypropylene group comprising up to 10, preferably up to 5, oxypropylene units. In one preferred embodiment, the invention relates to an acid stabilized human insulin derivative in which the B30 amino acid residue is deleted or is any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys; the A21 and the B3 amino acid residues are, independently, any amino acid residues which can be coded for by the genetic code except Lys, Arg and Cys; Phe^B1 may be deleted; the epsilon-amino group of Lys^B29 has a lipophilic substituent which comprises at least 6 carbon atoms; and 2-A Zn²⁺ ions may be bound to each insulin hexamer with the proviso that when B30 is Thr or Ala and A21 and B3 are both Asn, and Phe^B1 is not deleted, then 2-A Zn²⁺ ions are bound to each hexamer of the insulin derivative.

In another embodiment, the invention relates to an acid stabilized human insulin derivative in which the B30 amino acid residue is deleted or is any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys; the A21 and the B3 amino acid residues are, independently, any amino acid residues which can be coded for by the genetic code except Lys, Arg and Cys, with the proviso that if the B30 amino acid residue is Ala or Thr, then at least one of the residues A21 and B3 is different from Asn; Phe^B1 may be deleted; and the epsilon-amino group of Lys^B29 has a lipophilic substituent which comprises at least 6 carbon atoms.

In another embodiment, the invention relates to an acid stabilized human insulin derivative in which the B30 amino acid residue is deleted or is any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys; the A21 and the B3 amino acid residues are, independently, any amino acid residues which can be coded for by the genetic code except Lys, Arg and Cys; Phe^B1 may be deleted; the epsilon-amino group of Lys^B29 has a lipophilic substituent which comprises at least 6 carbon atoms; and 2-4 Zn²⁺ ions are bound to each insulin hexamer.

In one embodiment, the formulations of the invention contain acid stabilized insulin in a concentration from about 0.01 mM to about 2.OmM.

In another embodiment, the formulations of the invention contain acid stabilized insulin in a concentration from about 0.01 mM to about 1.OmM.

In yet another embodiment, the formulations of the invention contain acid stabilized insulin in a concentration from about 0.01 mM to about 0.5mM. In yet another embodiment, the formulations of the invention contain acid stabilized insulin in a concentration from about 0.01 mM to about 0.3mM. In the formulations of the invention, the salt of protamine or other positively charged peptides to be included is to be a protamine salt other than protamine sulphate or a salt of other positively charged peptides where such salts include, but are not limited to, acetate, bromide , chloride, caproate, trifluoroacetate, HCO₃ , propionate, lactate, formiate, nitrate, citrate, monohydrogenphosphate, dihydrogenphosphate, tartrate, or perchlorate salts or mixtures of any two salts.

In one embodiment, the salt of protamine or other positively charged peptides is selected from the group consisting of propionate, lactate, formiate, nitrate, acetate, citrate, caproate, monohydrogenphosphate, dihydrogenphosphate salts. In another embodiment, the salt of protamine or other positively charged peptides is selected from the group consisting of propionate, lactate, formiate, nitrate and acetate salts. In another embodiment, the salt of protamine or other positively charged peptides is selected from acetate salts.

In a further embodiment, when the salt of protamine or other positively charged pep- tides to be included in the formulation of the invention is to be a mixture of two different salts, one salt will be acetate and the other salt is selected from the group consisting of propionate, lactate, formiate, and nitrate salts. It is to be understood that when the salt to be included in the formulation of the invention is to be a mixture of two different salts, the molar ratio between the two different salts may be from 0.1 :1 to 1 :1. "Protamine" as used herein refers to the generic name of a group of strongly basic proteins present in sperm cells in salt-like combination with nucleic acids. Normally, protamines to be used together with insulin are obtained from e.g. salmon (salmine), rainbow trout (iridine), herring (clupeine), sturgeon (sturine), orspanish mackerel or tuna (thynnine) and a wide variety of salts of protamines are commercially available. Of course, it is under- stood that the peptide composition of a specific protamine may vary depending of which family, genera or species of fish it is obtained from. Protamine usually contains four major components, i.e. single-chain peptides containing about 30-32 residues of which about 21-22 are arginines. The N-terminal is proline for each of the four main components, and since no other amino groups are present in the sequence, chemical modification of protamine by a particu- lar salt is expected to be homogenous in this context.

In one embodiment, the protamine salts used in the present invention are from salmon.

In another embodiment, the protamine salts used in the present invention are from herring. In another embodiment, the protamine salts used in the present invention are from rainbow trout. In another embodiment, the protamine salts used in the present invention are from tuna.

"Positively charged peptides" as used herein refers to peptides of which the net charge at near neutral pH and below that is positive. In one embodiment, positively charges peptides used in the present invention are peptides with at net charge at near neutral pH and below that is greater than or equal to +2 and a percentage of positively charged amino acids that is greater than or equal to 40%.

In one embodiment, positively charges peptides used in the present invention are peptides with at net charge at near neutral pH and below that is greater than or equal to +4 and a percentage of positively charged amino acids that is greater than or equal to 40%.

In one embodiment, positively charges peptides used in the present invention are peptides with at net charge at near neutral pH and below that is greater than or equal to +6 and a percentage of positively charged amino acids that is greater than or equal to 40%.

In one embodiment, positively charged peptides used in the present invention are peptides comprising Arg(R) and/or Lys(K) residues.

In another embodiment, positively charged peptides used in the present invention consist of Arginine(R) residues.

In another embodiment, positively charged peptides used in the present invention consist of Lysine(K) residues. In another embodiment, positively charged peptides used in the present invention consist of Lysine(K) and Arginine(R) residues.

In another embodiment, positively charged peptides used in the present invention equals parts of protamine sequences.

Examples of positively charged peptides are: Arg_X: where X is from the following list (2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16) Lys_x, where X is from the following list (2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16), and combinations thereof; and peptides that equals parts of protamine sequences exemplified by VSR₆G₂R₄.

Examples of specific positively charged peptides are: R₁, R₂, R₃, R₄, R₅, R₆, R₇, Rs, Rg, R₁0, R₁₁, Ri₂, R₁₃, Ri₄, Ri5, R₁6, K₁, K₂, K₃, K₄, K₅, K₆, K₇, K₈, K₉, K₁₀, K₁₁, K₁₂, K₁₃, K₁₄, K₁₅, K₁₆

In one embodiment, the molar ratio of salt of protamine or other positively charged peptides to acid stabilized insulin in the formulations of the invention is from about 0.01 to about 2.

In another embodiment, the molar ratio of salt of protamine or other positively charged peptides to acid stabilized insulin in the formulations of the invention is from about 0.01 to about 1. In another embodiment of the invention, the formulation has a pH less than about 5.0 where the term "about" as used in connection with pH means + or - 0.1 pH units from the stated number.

In a further embodiment of the invention, the formulation has a pH less than about 4.5 .

In a further embodiment of the invention, the formulation has a pH less than about 4.2 .

It has been observed that the pH of the formulations of the invention is quite stable in that only very minor pH-migrations in the formulations of the invention have been observed to occur over time (data not shown) and that these variations are typically the pH-meter to pH-meter variations that are normally observed in measuring the pH of formulations.

In another embodiment of the invention, the formulations contain, in addition to an acid stabilized insulin and a salt of protamine or other positively charged peptides, at least one of the following components: a preservative, a divalent metal ion such as zinc, and an isotonicity agent.

In another embodiment of the invention, the formulations contain, in addition to an acid stabilized insulin and a salt of protamine or other positively charged peptides, at least two of the following components: a preservative, a divalent metal ion such as zinc, and an isotonicity agent. In another embodiment of the invention, the formulations contain, in addition to an acid stabilized insulin and a salt of protamine or other positively charged peptides, all three of the following components: a preservative, a divalent metal ion such as zinc, and an isotonicity agent.

In another embodiment of the invention, the formulation contains no divalent metal ion.

Where a pharmaceutically acceptable preservative is to be included in the formulations of the invention, the preservative is selected from the group consisting of phenol, tricresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p- hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, or mixtures thereof. Each one of these specific preservatives constitutes an alternative embodiment of the invention. In a preferred embodiment of the invention the preservative is phenol or m-cresol. In another preferred embodiment of the invention the preservative is phenol. In another preferred embodiment of the invention the preservative is m-cresol.

In a further embodiment of the invention the preservative is present in a concentra- tion from about 0.1 mg/ml to about 50 mg/ml, more preferably in a concentration from about 0.1 mg/ml to about 25 mg/ml, and most preferably in a concentration from about 0.1 mg/ml to about 10 mg/ml.

The use of a preservative in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice ofPhar- macy, 19^th edition, 1995.

Where a divalent metal ion is to be included in the formulations of the invention, the divalent metal ion may be calcium, magnesium or zinc or a combination thereof.

In one embodiment, the divalent metal ion is zinc.

In another embodiment, the concentration of zinc in the formulations of the invention is less than a molar ratio of 3 Zn²⁺ per insulin.

In another embodiment, the concentration of zinc in the formulations of the invention is less than a molar ratio of 2 Zn²⁺ per insulin.

In another embodiment, the concentratjon of zinc in the formulations of the invention is less than a molar ratio of 1 Zn²⁺ per insulin. Where a pharmaceutically acceptable isotonicity agent is to be included in the formulations of the invention, the isotonicity agent may be selected from the group consisting of glycerol, mannitol, propylene glycol, dimethyl sulfone, methyl sulfonyl methane, trehalose, sucrose, sorbitol, saccarose and/or lactose or mixtures thereof. In a preferred embodiment of the invention the isotonicity agent is glycerol. In a further embodiment of the invention the isotonicity agent is present in a concentration from about 0.5% to about 3%, more preferably in a concentration from about 1 % to about 2%, and most preferably in a concentration from about 1.6%.

The use of an isotonicity agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19^th edition, 1995.

In another embodiment of the invention, a buffer may be included in the formulations of the invention.

Where a buffer is to be included in the formulations of the invention, the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylgly- cine, histidine, glycine, lysine, arginin, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan, or mixtures thereof. Each one of these specific buffers constitutes an alternative embodiment of the invention. In a preferred embodiment of the invention the buffer is glycylglycine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate or mixtures thereof. In a further embodiment of the invention the formulation may further comprise a stabiliser selected from the group of high molecular weight polymers or low molecular compounds where such stabilizers include, but are not limited to, polyethylene glycol (e.g. PEG 3350), polyvinylalcohol (PVA), polyvinylpyrrolidone, carboxymethylcellulose, different salts (e.g. sodium chloride), L-glycine, L-histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine and mixtures thereof. Each one of these specific stabi- lizers constitutes an alternative embodiment of the invention. In a preferred embodiment of the invention the stabiliser is selected from the group consisting of L-histidine, imidazole and arginine.

In a further embodiment of the invention the high molecular weight polymer is present in a concentration from 0.1 mg/ml to 50mg/ml. In a further embodiment of the invention the high molecular weight polymer is present in a concentration from 0.1 mg/ml to 5mg/ml. In a further embodiment of the invention the high molecular weight polymer is present in a concentration from 5mg/ml to 10mg/ml. In a further embodiment of the invention the high molecular weight polymer is present in a concentration from 0mg/ml to 20mg/ml. In a further embodiment of the invention the high molecular weight polymer is present in a concentration from 20mg/ml to 30mg/ml. In a further embodiment of the invention the high molecular weight polymer is present in a concentration from 30mg/ml to 50mg/ml.

In a further embodiment of the invention the low molecular weight compound is present in a concentration from 0.1 mg/ml to 50mg/ml. In a further embodiment of the invention the low molecular weight compound is present in a concentration from 0.1 mg/ml to 5mg/ml. In a further embodiment of the invention the low molecular weight compound is present in a concentration from 5mg/ml to 10mg/ml. In a further embodiment of the invention the low molecular weight compound is present in a concentration from 10mg/ml to 20mg/ml. In a further embodiment of the invention the low molecular weight compound is present in a concentration from 20mg/ml to 30mg/ml. In a further embodiment of the invention the low mo- lecular weight compound is present in a concentration from 30mg/ml to 50mg/ml.

The use of a stabilizer in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19^th edition, 1995.

In a further embodiment of the invention the formulation of the invention may further comprise a surfactant where a surfactant may be selected from a detergent, ethoxylated castor oil, polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fatty acid esters, polysorbate, such as polysorbate-20, block copolymers as polyethyleneoxide/polypropylene- oxide block copolymers such as poloxamers, poloxamer 188 and poloxamer 407, Brij^® 35, Brij^® 56, Brij^® 72, Brij^® 76, Brij^® 92V, Brij^® 97, Brij^® 58P, Cremophor^® EL, Decaethylene glycol monododecyl ether, N-Decanoyl-N-methylglucamine, n-Dodecanoyl-N-methylglucamide, alkyl-polyglucosides, ethoxylated castor oil, Heptaethylene glycol monodecyl ether, Heptaethylene glycol monododecyl ether, Heptaethylene glycol monotetradecyl ether, Hexaethylene glycol monododecyl ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol monooctadecyl ether, Hexaethylene glycol monotetradecyl ether, lgepal CA-630, lgepal CA-630, Methyl-6-O-(N-heptylcarbamoyl)-beta-D-glucopyranoside, Nona- ethylene glycol monododecyl ether, N-Nonanoyl-N-methylglucamine, N-Nonanoyl-N- methylglucamine, Octaethylene glycol monodecyl ether, Octaethylene glycol monododecyl ether, Octaethylene glycol monohexadecyl ether, Octaethylene glycol monooctadecyl ether, Octaethylene glycol monotetradecyl ether, Octyl-β-D-glucopyranoside, Pentaethylene glycol monodecyl ether, Pentaethylene glycol monododecyl ether, Pentaethylene glycol monohexa- decyl ether, Pentaethylene glycol monohexyl ether, Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol monooctyl ether, Polyethylene glycol diglycidyl ether, Polyethylene glycol ether W-1 , Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate, PoIy- oxyethylene 20 isohexadecyl ether, Polyoxyethylene 20 oleyl ether, Polyoxyethylene 40 stearate, Polyoxyethylene 50 stearate, Polyoxyethylene 8 stearate, Polyoxyethylene bis(im- idazolyl carbonyl), Polyoxyethylene 25 propylene glycol stearate, Saponin from Quillaja bark, Span^® 20, Span^® 40, Span^® 60, Span^® 65, Span^® 80, Span^® 85, Tergitol, Type 15-S-12, Tergitol, Type 15-S-30, Tergitol, Type 15-S-5, Tergitol, Type 15-S-7, Tergitol, Type 15-S-9, Tergitol, Type NP-10, Tergitol, Type NP-4, Tergitol, Type NP-40, Tergitol, Type NP-7, Tergitol, Type NP-9, Tetradecyl-β-D-maltoside, Tetraethylene glycol monodecyl ether, Tetraethylene glycol monododecyl ether, Tetraethylene glycol monotetradecyl ether, Tri- ethylene glycol monodecyl ether, Triethylene glycol monododecyl ether, Triethylene glycol monohexadecyl ether, Triethylene glycol monooctyl ether, Triethylene glycol monotetradecyl ether, Triton CF-21 , Triton CF-32, Triton DF-12, Triton DF-16, Triton GR-5M, Triton QS-15, Triton QS-44, Triton X-100, Triton X-102, Triton X-15, Triton X-151 , Triton X-200, Triton X- 207, Triton^® X-100, Triton^® X-114, Triton^® X-165 solution, Triton^® X-305 solution, Triton^® X- 405, Triton^® X-45, Triton^® X-705-70, TWEEN^® 20, TWEEN^® 40, TWEEN^® 60, TWEEN^® 6, TWEEN^® 65, TWEEN^® 80, TWEEN^® 81 , TWEEN^® 85, Tyloxapol,, glycerol, cholic acid or derivatives thereof, lecithins, alcohols and phospholipids, glycerolphospholipids (lecithins, kephalins, phosphatidyl serine), glycerolglycolipids (galactopyransoide), sphingophospho- lipids (sphingomyelin), and sphingoglycollipids (ceramides, gangliosides), DSS (docusate sodium, docusate calcium, docusate potassium, SDS (sodium dodecyl sulfate or sodium lauryl sulfate), dipalmitoyl phosphatidic acid, sodium caprylate, bile acids and salts thereof and glycine or taurine conjugates, ursodeoxycholic acid, sodium cholate, sodium deoxy- cholate, sodium taurocholate, sodium glycolcholate, N-Hexadecyl-N,N-dimethyl-3-ammonio- 1-propanesulfonate, anionic (alkyl-aryl-sulphonates) monovalent surfactants, palmitoyl lysophosphatidyl-L-serine, lysophospholipids (e.g. 1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine or threonine), alkyl, alkoxyl (alkyl ester), alkoxy (alkyl ether)- derivatives of lysophosphatidyl and phosphateidylcholines, e.g. lauroyl and myristoyl derivatives of lysophosphatidylcholine, dipalmitoylphosphatidylcholine, and modifications of the polar head group, that is cholines, ethanolamines, phosphatidic acid, serines, threonines, glycerol, inositol, and the positively charged DODAC, DOTMA, DCP, BISHOP, lysophosphat- idylserine and lysophosphatidylthreonine, zwitterionic surfactants (e.g. N-alkyl-N,N-dimethyl- ammonio-1 -propanesulfonates, 3-cholamido-1 -propyldimethylammonio-1 -propanesulfonate, dodecylphosphocholine, myristoyl lysophosphatidylcholine, hen egg lysolecithin), cationic surfactants (quarternary ammonium bases) (e.g. cetyl-trimethylammonium bromide, cetyl- pyridinium chloride), non-ionic surfactants, polyethyleneoxide/polypropyleneoxide block copolymers (Pluronics/Tetronics, Triton X-100, Dodecyl β-D-glucopyranoside) or polymeric surfactants (TWEEN^® 40, TWEEN^® 80, Brij-35), fusidic acid derivatives- (e.g. sodium tauro- dihydrofusidate etc.), long-chain fatty acids and salts thereof C6-C12 (eg. oleic acid and caprylic acid), acylcarnitines and derivatives, N^α-acylated derivatives of lysine, arginine or histidine, or side-chain acylated derivatives of lysine or arginine, N^α-acylated derivatives of dipeptides comprising any combination of lysine, arginine or histidine and a neutral or acidic amino acid, N^α-acylated derivative of a tripeptide comprising any combination of a neutral amino acid and two charged amino acids, or the surfactant may be selected from the group of imidazoline derivatives, or mixtures thereof. Each one of these specific surfactants constitutes an alternative embodiment of the invention. In a preferred embodiment of the invention the surfactant is poloxamer 188 or TWEEN^® 20. In another preferred embodiment of the invention the surfactant is poloxamer 188. In another preferred embodiment of the invention the surfactant is TWEEN^® 20.

In a further embodiment of the invention the surfactant is present in an amount less that 200ppm, more preferably in an amount less that 100ppm, and most preferably in an amount less that 50ppm.

The use of a surfactant in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19^th edition, 1995. The formulations of the invention may be prepared by conventional techniques, e.g. as described in Remington's Pharmaceutical Sciences, 1985 or in Remington: The Science and Practice of Pharmacy, 19^th edition, 1995, where such conventional techniques of the pharmaceutical industry involve dissolving and mixing the ingredients as appropriate to give the desired end product. The present invention also relates to methods of making the formulations of the invention. In one embodiment, the method for preparing formulations of the invention comprises: a) preparing a solution by dissolving a divalent metal ion in water or buffer; b) preparing a solution by dissolving the preservative in water or buffer; c) preparing a solution by dissolving the isotonicity agent in water and buffer d) preparing a solution by dissolving the surfactant in water or buffer; e) preparing a solution by dissolving the acid stabilized insulin in water or buffer; f) preparing a solution by dissolving a salt of protamine or other positively charged peptides in buffer or water; g) mixing solution e) and one or more of solutions a), b), c), and d); h) mixing solution g) with solution f); and i) adjusting the pH of the mixture in h)l to the desired pH of less than 5.0.

In another embodiment, a solution containing acid stabilized insulin and optionally a preservative, an isotonicity agent and/or a divalent metal ion in water or a buffer at a pH of about 6.5 to about 7.5, preferably at about neutral pH, can be mixed with a solution of a salt of protamine or other positively charged peptides, and then the pH of the mixed solution can be adjusted to the desired final pH of less than 5.0.

Where the formulations of the invention contain mixtures of salts of protamine or other positively charged peptides, the solution of the salt to be used in the above mixtures can be prepared by dissolving each salt separately and then mixing the solutions together or by dissolving the salts together in one volume of water or buffer.

Of course, it is to be understood the components of the final formulation may be mixed together in orders other than those set forth above so long as the final formulation reaches the same equilibrium state at the end of mixing. The present invention further relates to methods of treatment using the pharmaceutical formulations of the invention where the compositions are administered in an amount effective to combat the disease, condition, or disorder for which administration of the insulin peptide contained in the formulation is indicated.

In one embodiment, the formulations of the invention may be used in the treatment of type 1 and type 2 diabetes.

The dose, route of administration, and number of administrations per day of a formulation of the invention will be determined by a physician taking into account such factors as the therapeutic objectives, the nature and cause of the patient's disease, other drugs or medications the patient might be taking, the patient's gender and weight, level of exercise and eating habits as well as other factors that might be known to the physician. In a broad range, the daily dose of insulin to be administered to a patient in the formulations of the invention is from about 0.1 units of insulin/kg of body weight to about 1 unit of insulin/kg of body weight.

In another embodiment, the daily dose of insulin to be administered to a patient in the formulations of the invention is from about 0.2 units of insulin/kg of body weight to about 0.6 units of insulin/kg of body weight. Of course, the physician of ordinary skill in treating diabetes would understand that the concentration ranges of insulin used to treat a diabetic patient may vary depending on whether, for example, the patient to be treated is a child with type 1 diabetes or an adult with strongly insulin resistant type 2 diabetes. The physician of ordinary skill in treating diabetes will also be able to select the therapeutically most advantageous method for administering the formulations of the invention.

In one embodiment, the formulations may be administered parenterally where typical routes of parenteral administration are subcutaneous and intramuscular. In another embodiment, the formulations may be administered parenterally where the route is subcutane- ous.

In another embodiment, the formulations may be administered by nasal, buccal, pulmonary or ocular routes. In another embodiment, the formulations may be administered by nasal route. In another embodiment, the formulations may be administered by pulmonary route. In one embodiment the formulations of the invention are used in connection with insulin pumps. The insulin pumps may be prefilled and disposable, or the insulin formulations may be supplied from a reservoir which is removable. Insulin pumps may be skin-mounted or carried, and the path of the insulin preparation from the storage compartment of the pump to the patient may be more or less tortuous. Non-limiting examples of insulin pumps are dis- closed in US 5,957,895, US 5,858,001 , US 4,468,221 , US 4,468,221 , US 5,957,895, US 5,858,001 , US 6,074,369, US 5,858,001 , US 5,527,288, and US 6,074,369.

In another embodiment the formulations of the invention are used in connection with pen-like injection devices, which may be prefilled and disposable, or the insulin formulations may be supplied from a reservoir which is removable. Non-limiting examples of pen-like in- jection devices are FlexPen^®, InnoLet^®, InDuo™, Innovo^®.

In a further embodiment, formulations of the invention are used in connection with devices for pulmonary administration of aqueous insulin formulations, a non-limiting example of which is the AerX^® device.

The invention furthermore relates to treatment of a patient in which the pharmaceu- tical formulations of the invention are combined with another form of treatment. In one aspect of the invention, treatment of a patient with the pharmaceutical formulations of the invention is combined with diet and/or exercise.

In another aspect of the invention the pharmaceutical formulations of the invention are administered in combination with one or more further active substances in any suitable ratios where "in combination with" as used in connection with the pharmaceutical formulations of the invention and one or more further active substances means that the one or more further active substances may be included within the formulation of the invention or they may be contained in separate formulation(s) from the formulation of the invention. Such further active substances may e.g. be selected from antiobesity agents, antidiabetics, antihypertensive agents, agents for the treatment of complications resulting from or associated with diabetes and agents for the treatment of complications and disorders resulting from or associated with obesity.

Thus, in a further aspect of the invention the pharmaceutical formulations of the invention may be administered in combination with one or more antiobesity agents or appetite regulating agents. Such agents may be selected from the group consisting of CART (cocaine amphetamine regulated transcript) agonists, NPY (neuropeptide Y) antagonists, MC4 (melanocortin 4) agonists, MC3 (melanocortin 3) agonists, orexin antagonists, TNF (tumor necrosis factor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP (corticotropin releasing factor binding protein) antagonists, urocortin agonists, β3 adrenergic agonists such as CL-316243, AJ- 9677, GW-0604, LY362884, LY377267 or AZ^0140, MSH (melanocyte-stimulating hormone) agonists, MCH (melanocyte-concentrating hormone) antagonists, CCK (cholecystokinin) agonists, serotonin re-uptake inhibitors such as fluoxetine, seroxat or citalopram, serotonin and noradrenaline re-uptake inhibitors, mixed serotonin and noradrenergic compounds, 5HT (serotonin) agonists, bombesin agonists, galanin antagonists, growth hormone, growth factors such as prolactin or placental lactogen, growth hormone releasing compounds, TRH (thyreotropin releasing hormone) agonists, UCP 2 or 3 (uncoupling protein 2 or 3) modulators, leptin agonists, DA agonists (bromocriptin, doprexin), lipase/amylase inhibitors, PPAR (peroxisome proliferator- activated receptor) modulators, RXR (retinoid X receptor) modulators, TR β agonists, AGRP (Agouti related protein) inhibitors, H3 histamine antagonists, opioid antagonists (such as naltrexone), exendin^, GLP-1 , glp-1 analogues or derivatives hereof and ciliary neurotrophic factor.

In one embodiment of the invention the antiobesity agent is leptin.

In another embodiment the antiobesity agent is dexamphetamine or amphetamine.

In another embodiment the antiobesity agent is fenfluramine or dexfenfluramine. In still another embodiment the antiobesity agent is sibutramine.

In a further embodiment the antiobesity agent is orlistat. In another embodiment the antiobesity agent is mazindol or phentermine. In still another embodiment the antiobesity agent is phendimetrazine, diethylpropion, fluoxetine, bupropion, topiramate or ecopipam.

The orally active hypoglycemic agents comprise imidazolines, sulphonylureas, biguanides, meglitinides, oxadiazolidinediones, thiazolidinediones, insulin sensitizers, insulin secretagogues such as glimepride, α-glucosidase inhibitors, agents acting on the ATP- dependent potassium channel of the β-cells eg potassium channel openers such as those disclosed in WO 97/26265, WO 99/03861 and WO 00/37474 (Novo Nordisk A/S) which are incorporated herein by reference, or mitiglinide, or a potassium channel blocker, such as BTS-67582, nateglinide, glucagon antagonists such as those disclosed in WO 99/01423 and WO 00/39088 (Novo Nordisk A/S and Agouron Pharmaceuticals, Inc.), which are incorporated herein by reference, GLP-1 agonists such as those disclosed in WO 00/42026 (Novo Nordisk A/S and Agouron Pharmaceuticals, Inc.), which are incorporated herein by reference, DPP-IV (dipeptidyl peptidase-IV) inhibitors, PTPase (protein tyrosine phosphatase) in- hibitors, inhibitors of hepatic enzymes involved in stimulation of gluconeogenesis and/or gly- cogenolysis, glucose uptake modulators, GSK-3 (glycogen synthase kinase-3) inhibitors, compounds modifying the lipid metabolism such as antilipidemic agents, compounds lowering food intake, PPAR (peroxisome proliferator-activated receptor) and RXR (retinoid X receptor) agonists, such as ALRT-268, LG-1268 or LG-1069. In a further embodiment of the invention the pharmaceutical formulations of the invention are administered in combination with a sulphonylurea e.g. tolbutamide, chlorpropamide, tolazamide, glibenclamide, glipizide, glimepiride, glicazide or glyburide.

In another embodiment of the invention the pharmaceutical formulations of the invention are administered in combination with a biguanide, e.g. metformin. In yet another embodiment of the invention the pharmaceutical formulations of the invention are administered in combination with a meglitinide eg repaglinide or nateglinide.

In still another embodiment of the invention the pharmaceutical formulations of the invention are administered in combination with a thiazolidinedione insulin sensitizer, e.g. tro- glitazone, ciglitazone, pioglitazone, rosiglitazone, isaglitazone, darglitazone, englitazone, CS- 01 1/CI-1037 or T 174 or the compounds disclosed in WO 97/41097, WO 97/41 119, WO 97/41 120, WO 00/41 121 and WO 98/45292 (Dr. Reddy's Research Foundation), which are incorporated herein by reference.

In still another embodiment of the invention the pharmaceutical formulations of the invention may be administered in combination with an insulin sensitizer, e.g. such as Gl 262570, YM-440, MCC-555, JTT-501 , AR-H039242, KRP-297, GW-409544, CRE-16336, AR-H049020, LY510929, MBX-102, CLX-0940, GW-501516 or the compounds disclosed in WO 99/19313, WO 00/50414, WO 00/63191 , WO 00/63192, WO 00/63193 (Dr. Reddy's Research Foundation) and WO 00/23425, WO 00/23415, WO 00/23451 , WO 00/23445, WO 00/23417, WO 00/23416, WO 00/63153, WO 00/63196, WO 00/63209, WO 00/63190 and WO 00/63189 (Novo Nordisk A/S), which are incorporated herein by reference. In a further embodiment of the invention the pharmaceutical formulations of the invention are administered in combination with an α-glucosidase inhibitor, e.g. voglibose, emiglitate, miglitol or acarbose.

In another embodiment of the invention the pharmaceutical formulations of the invention are administered in combination with an agent acting on the ATP-dependent potas- sium channel of the β-cells, e.g. tolbutamide, glibenclamide, glipizide, glicazide, BTS-67582 or repaglinide.

In yet another embodiment of the invention the pharmaceutical formulations of the invention may be administered in combination with nateglinide.

In still another embodiment of the invention the pharmaceutical formulations of the invention are administered in combination with an antilipidemic agent, e.g. cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol or dextrothyro- xine.

In another aspect of the invention, the pharmaceutical formulations of the invention are administered in combination with more than one of the above-mentioned compounds, e.g. in combination with metformin and a sulphonylurea such as glyburide; a sulphonylurea and acarbose; nateglinide and metformin; acarbose and metformin; a sulphonylurea, metformin and troglitazone; metformin and a sulphonylurea; etc.

Furthermore, the pharmaceutical formulations of the invention may be administered in combination with one or more antihypertensive agents. Examples of antihypertensive agents are β-blockers such as alprenolol, atenolol, timolol, pindolol, propranolol and metoprolol, ACE (angiotensin converting enzyme) inhibitors such as benazepril, captopril, enalapril, fosinopril, lisinopril, quinapril and ramipril, calcium channel blockers such as nifedipine, felodipine, nicer- dipine, isradipine, nimodipine, diltiazem and verapamil, and α-blockers such as doxazosin, urapidil, prazosin and terazosin. The pharmaceutical preparation of the invention may also be combined with NEP inhibitors such as candoxatril.

Further reference can be made to Remington: The Science and Practice of Pharmacy, 19^th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995.

It should be understood that any suitable combination of the compounds according to the invention with diet and/or exercise, one or more of the above-mentioned compounds and optionally one or more other active substances are considered to be within the scope of the present invention. All scientific publications and patents cited herein are specifically incorporated by reference. The following examples illustrate various aspects of the invention but are in no way intended to limit the scope thereof.

EXAMPLES

EXAMPLE 1

Equilibrium Solubility of protamine acetate-containing insulin formulations

To a standard formulation of 0.6mM insulin, 0.3mM Zn2+, 3OmM phenol, 1.6% glycerol, 1OmM Tris, pH=7.4 varying amounts of protamine acetate was added, and 0.3 ml sam- pies were incubated at 23 ⁰C for at least 4 days. After centrifugation (20,000 g for 20 minutes at 23 C) of each sample, pH was measured and the solubility was determined by quantification of insulin contents in the supernatant by reverse phase HPLC on a Xterra RP8 Guard C8 column, 3,9 x 20mm ID, 5 micron particle size, eluted at 1 ml/minute at 35⁰C. The monomeric insulin compounds are eluted isocratically/gradiently with a phosphate buffer, pH 7,2 contain- ing sodium sulphate and approximately 7,7% (W/W) acetonitrile. - 2 minutes isocratically 20% buffer B (65,5% acetonitrile) followed by 1 min gradient step with increased acetonitrile concentration, 20 to 50% buffer B, for elution of the more hydrophobic compounds.

Figure 1 shows the solubility of human insulin, B28D insulin and A21G, B28D, desB30 insulin in standard formulation as described with different concentrations of pro- tamine acetate. In this standard formulation of 0.6mM insulin the solubility of insulin at neutral pH is gradually reduced upon addition of protamine acetate leading to a complete precipitation of insulin at a protamine acetate concentration of 0.3mM.

EXAMPLE 2 General solid phase procedure for preparation of peptides

m is 0-4 and n is 1-20.

Solid phase peptide synthesis protocol (I):

Fmoc-removal: The resin was treated with piperidine/NMP/DBU 20/80/2 for a period of 10 min, drained and again retreated as above for 2h.

Washing: 6 times with NMP

Coupling: A mixture of Fmoc-Arg(Pbf)-OH (3 eq) + HOAt (3 eq) in NMP was preactivated with DIC (3 eq) for 10 min, the mixture was then added to the deprotected resin and the slurry shaken gently for 3-16 h. The resin was then dried and a new coupling was carried out using the same amounts as above, reaction time was 3-16 h.

Capping using 10 eq of activated AcOH: A mixture of AcOH (10 eq) + HOBt (10 eq) dissolved in NMP was added to the resin followed by DIC (10 eq). Reaction time was 1-2 h.

Washing: 6 times with NMP

H-Arα6-NH2.TFA

Fmoc-protected Rink amide AM resin (NovaBiochem, 0.68 mmol/g, 14.7 g, 10 mmol) was used to prepare resin bound Fmoc-(Arg(Pbf))₆ by the solid phase peptide synthesis protocol (I). The following amounts were used for each coupling: Fmoc-Arg(Pbf)-OH 19,44 g (30 mmol), HOAt 4.04 g (30 mmol) and DIC 4,67 ml. (30 mmol) in NMP (50 ml_). Capping: AcOH 3.36 ml_, HOBt 7.93 g and DIC12.0 ml. in NMP (25 ml_). De-Fmoc conditions: As in protocol (I).

After 6 double couplings the dried resin was washed with DCM x 2 followed by wash with ether x 2. Drying overnight resulted in 45 g resin bound Fmoc-(Arg(Pbf))₆.

1O g of this was deprotected using the general procedure washed and dried and then treated with TFA/TIS/EtOH (200 ml.) for 5 h, evaporated to 20 ml. which was slowly added to cold ether (200 ml.) under nitrogen resulting in formation of a precipitate. This was washed 3 times with ether and dried in vacuo. Yield 2.2 g.

This product was dissolved in HAc (1 M), freeze-dried and subsequently purified on Gil- son HPLC on a C18 column ( Jones, Kromasil RP18 5μm 15x225 mm ) with acetonitrile/water /0.1 % TFA as eluent. The isolated product was freeze dried resulting in 717 mg of the TFA salt of arg6-NH2, LCMS: Rt.: 2.96 min, m/z: 954; MALDI-MS : m/z 954.

Arα8-NH2.TFA

This compound was prepared from the Fmoc-(Arg(Pbf))₆ derivative above, adding two more double couplings. Cleavage and purification as above. Praep Gilson HPLC; Column: Jones, Kromasil RP18 5μm 15x225 mm: Rt = 7.5 min, Flow:10 ml/ min, Eluent:: water/ acetonitril/0,1% TFA. Gradient: 0 -1 min: 10 % CH3CN, 1-20 min:10 % CH3CN to 50 % CH3CN, 20-25 min 50 % CH3CN. MALDI: (matrix CHCA); m/z:1267. LCMS: Rt.: 3.03 min, m/z:634 ( MW/2).

H-Arα-12-NH2.TFA

This compound was prepared from the Fmoc-(Arg(Pbf))₆ derivative above adding 6 more double couplings. Cleavage and purification as above.

Praep Gilson HPLC; Column: Jones, Kromasil RP18 5μm 15x225 mm: Rt. 7-8 min,

Flow: 10 ml_/ min, Gradient: 0-5 min: 5 % CH3CN, 5 -20 min: 5% CH3CN to 50 % CH3CN.

MALDI- MS: m/z 1887.6 (Matrix: Sinnapinic acid).

Val-Ser-Arα6-Glv2-Arα4.TFA

Preparation from Novabiochem H-Arg(Pbf)-2-CITrt-resin (0.72 mmol/g, 5g, 3.6 mmol) using the standard technology described above and coupling with 3 times Fmoc-Arg(Pbf)-OH, 2 times Fmoc-Gly-OH, 6 times Fmoc-Arg(Pbf)-OH, 1 x Fmoc-Ser(OtBu)-OH and finally 1 x Fmoc- VaI-OH resulted in 10.7 g crude resin-bound product.

Cleavage with TFA/TIS/EtOH for 7 h followed by evaporation to 20 mL and pouring into cold ether resulted in 4.25 g crude precipitate. LCMS: Rt.:1.11 min; m/z: 628 correspond- ing to MW/3. Purification on praep. Gilson HPLC gave totally 500 mg of pure product. Column: Jones, Kromasil RP18 5μm 15x225 mm: Rt. 8-9min, Flow: 10 mL/ min, Gradient: 0-5 min: 5 % CH3CN / 0.1 % HAc, 5 -20 min: 5% to 50 % CH3CN / 0.1 % HAc. MALDI- MS: m/z 1882.5 (Matrix: CHCA). LCMS: Rt.: 1.15 min, m/z: 628 and 941 corresponding to MW/2 and MW/3.

While the invention has been described and illustrated with reference to certain pre- ferred embodiments thereof, those skilled in the art will appreciate that various changes, modifications, and substitutions can be made therein without departing from the spirit and scope of the present invention. For example, effective dosages other than the preferred dosages as set forth herein may be applicable as a consequence of variations in the responsiveness of the mammal being treated for the disease(s). Likewise, the specific pharmacological responses observed may vary according to and depending on the particular active compound selected or whether there are present pharmaceutical carriers, as well as the type of formulation and mode of administration employed, and such expected variations or differences in the results are contemplated in accordance with the objects and practices of the present invention.

Accordingly, the invention is not to be limited as by the appended claims. The features disclosed in the foregoing description and/or in the claims may both separately and in any combination thereof be material for realising the invention in diverse forms thereof.

Preferred features of the invention:

1. A pharmaceutical formulation which is a solution comprising an acid stabilized insulin, and a salt of protamine or other positively charged peptides, wherein said salt is present in said formulation in a concentration of greater than 0.01 mM and wherein said formulation has a pH of less than about 5.0.

2. The formulation according to clause 1 , wherein the molar ratio of the salt of protamine or other positively charged peptides to acid stabilized insulin is from about 0.01 to about 2.

3. The formulation according to clause 2, wherein the molar ratio of the salt of protamine or other positively charged peptides to acid stabilized insulin is from about 0.01 to about 1.5.

4. The formulation according to clause 3, wherein the molar ratio of the salt of protamine or other positively charged peptides to acid stabilized insulin is from about 0.01 to 1.0.

5. The formulation according to any of the clauses 1-4, wherein the pH of said formulation is from about 3.0 to about 5.5. 6. The formulation according to clause 5, wherein the pH of said formulation is from about 3.0 to about 5.0.

7. The formulation according to clause 6, wherein the pH of said formulation is from about 3.0 to about 4.2.

8. The formulation according to any of the clauses 1-7, further comprising a preservative.

9. The formulation according to clause 8, wherein the preservative is m-cresol.

10 . The formulation according to any of the clauses 1-9, further comprising an isotonicity agent.

11. The formulation according to clause 10, wherein the isotonicity agent is glycerol.

12. The formulation according to any of the clauses 1-1 1 , further comprising a divalent metal ion.

13. The formulation according to clause 12, wherein the divalent metal ion is zinc.

14. The formulation according to any of the clauses 1-13, further comprising a surfactant.

15. The formulation according to clause 14, wherein the surfactant is TWEEN^® 20.

16. The formulation according to any of the clauses 1-15, wherein said acid stabilized insulin is A21G, desB30 human insulin.

17. The formulation according to any of the clauses 1-15, wherein said acid stabilized insulin is A21G, B28D, desB30 human insulin.

18. The formulation according to any of the clauses 1-15, wherein said acid stabilized insulin is A21G, B28E, desB30 human insulin.

19. A method of treating type 1 or type 2 diabetes, said method comprising administering to a patient in need of such treatment an effective amount of a formulation according to any of the clauses 1-18. 20. The formulation according to any of the clauses 1-18, wherein the salt of protamine or other positively charged peptides is selected from the group consisting of propionate, lactate, formiate, nitrate and acetate salts.

21. The formulation according to clause 20, wherein the salt is acetate.

22. The formulation according to any of the clauses 20-21 , further comprising a divalent metal ion.

23. The formulation according to any of the clauses 20-22, further comprising a preservative.

24. The formulation according to any of the clauses 20-23, further comprising an isotonicity agent.

25. The formulation according to any of the clauses 20-24, further comprising a surfactant.

26. The formulation according to any of the clauses 1-18, 20-25, wherein said formulation is soluble at a pH of less than about 5.0.

27. A method of treating type 1 or type 2 diabetes, said method comprising administering to a patient in need of such treatment an effective amount of a formulation according to any of the clausems 20-26.

28. The use of a formulation according to any of the clauses 1-18, 20-26 for the preparation of a formulation for treating type 1 or type 2 diabetes.

Claims

2. The formulation according to claim 1 , wherein the molar ratio of the salt of protamine or other positively charged peptides to acid stabilized insulin is from about 0.01 to about 2.

3. The formulation according to claim 2, wherein the molar ratio of the salt of protamine or other positively charged peptides to acid stabilized insulin is from about 0.01 to about 1.5.

4. The formulation according to claim 3, wherein the molar ratio of the salt of protamine or other positively charged peptides to acid stabilized insulin is from about 0.01 to 1.0.

5. The formulation according to any of the claims 1-4, wherein the pH of said formulation is from about 3.0 to about 5.5.

6. The formulation according to any of the claims 1-5, further comprising a preservative.

7 . The formulation according to any of the claims 1-6, further comprising an isotonicity agent.

8. The formulation according to any of the claims 1-7, further comprising a divalent metal ion.

9. The formulation according to any of the claims 1-8, further comprising a surfactant.

10. The formulation according to any of the claims 1-9, wherein said acid stabilized insulin is A21G, desB30 human insulin.

11. The formulation according to any of the claims 1-9, wherein said acid stabilized insulin is A21G, B28D, desB30 human insulin.

12. The formulation according to any of the claims 1-9, wherein said acid stabilized insulin is A21G, B28E, desB30 human insulin.

13. The formulation according to any of the claims 1-12, wherein the salt of protamine or other positively charged peptides is selected from the group consisting of propionate, lactate, formiate, nitrate and acetate salts.

14. The formulation according to any of the claims 1-13, wherein said formulation is soluble at a pH of less than about 5.0.

15. The use of a formulation according to any of the claims 1-13 for the preparation of a formulation for treating type 1 or type 2 diabetes.